Antibodies specific for tumor-associated antigens are a valuable approach in cancer therapy because they mediate selective destruction of tumor cells, while leaving healthy cells and tissues undamaged.
Members of the ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members, including epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185“e”), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). HER2 is a transmembrane surface-bound receptor tyrosine kinase and is normally involved in the signal transduction pathways leading to cell growth and differentiation. HER2 is a promising target for treatment of breast cancer as it was found to be overexpressed in about one-quarter of breast cancer patients (Bange et al, 2001, Nature Medicine 7:548).
The murine monoclonal antibody 4D5 is targeting HER2 specifically in HER2 overexpressing cancer cells, while having no effects on cells expressing physiological levels of her2. The humanized (4D5) monoclonal antibody (hu4D5) is commercially known as the drug Herceptin® (trastuzumab, rhuMAb HER2, U.S. Pat. No. 5,821,337), which gained FDA marketing approval in late 1998.
Herceptin was the first monoclonal antibody developed for the treatment of HER2-positive breast cancer and has increased survival times for patients so that they are now the same as for patients with HER2-negative breast cancer. Before Herceptin treatment, shorter survival outcomes were expected for patients diagnosed with HER2-positive breast cancer, compared to patients with HER2-negative disease. In the CLEOPATRA study, PERJETA in combination with Herceptin and chemotherapy has shown the extension of survival times for patients with this aggressive disease even further than Herceptin.
Pertuzumab (PERJETA®, rhuMab 2C4, U.S. Pat. No. 7,862,817) is a humanized monoclonal antibody, which is designed specifically to prevent the HER2 receptor from pairing (dimerising) with other HER receptors (EGFR/HER1, HER3 and HER4) on the surface of cells, a process that is believed to play a role in tumor growth and survival. The combination of PERJETA, Herceptin and chemotherapy is thought to provide a more comprehensive blockade of HER signaling pathways. PERJETA is approved in combination with Herceptin (trastuzumab) and docetaxel in adult patients with HER2-positive metastatic or locally recurrent unresectable breast cancer and gained FDA approval for neoadjuvant breast cancer treatment in September 2013. Pertuzumab binds to domain II of HER2, essential for dimerization, while Ttrastuzumab binds to extracellular domain IV of HER2.
Li et al (Cancer Research. 2013) describe bispecific, bivalent antibodies to ErbB2 that overcome trastuzumab resistance. The bispecific, bivalent antibodies described therein are based on the native Trastuzumab and Pertuzumab sequences.
Surprisingly the inventors of the present application found that optimizing the native Trastuzumab and Pertuzumab sequences and combining these optimized variants in two different improved bispecific, monovalent antibody formats leads to improved properties as compared to the combination of the monospecific antibodies rhuMab 2C4 and hu 4D5. Further the antibodies are superior to the bivalent antibody formats disclosed in Li et al, as they are monovalent and have the same molecular weight as the two monospecific antibodies Pertuzumab and Trastuzumab. Hence the new bispecific format combines the superior characteristics of the bispecific HER2 antibodies known in the art with the advantages of a classical monospecific antibody: The novel bispecific HER2 antibodies of the present invention are monovalent for the two different HER2 epitopes, resulting in the same avidity effect as the bivalent parental antibodies. In contrast, tetravalent antibodies may differ in their avidity for HER2 on cells. The avidity effect of the novel bispecific HER2 antibodies may result in a superior safety window on cell types with low HER2 expression such as in normal tissues or cardiac tissues where inhibition of HER2 and/or ADCC may not be desired.
Furthermore, the bispecific antibodies described herein have the same diffusion constants as the bivalent parental antibodies due to their natural IgG architecture that matches to the diffusion constant of the parental 150KD IgG1 antibody. Due to the natural IgG architecture furthermore the risk for immunogenicity and the formation of anti-drug antibodies can be expected to be reduced. Last but not least as compared to tetravalent bispecific antibodies the risk for the formation of immune complexes with shed HER2 extracellular domain is reduced by being monovalent and comparable to the parental antibodies. Immune complexes may result in the enhanced immunogenicity of the complex taken up by antigen presenting cells and ultimately can induce kidney toxicity if immune complexes are deposited in the kidney.
In one aspect of the invention a monovalent bispecific antibody is provided, wherein one of the Fab fragments of an IgG molecule is replaced by a crossover Fab fragment. Crossover Fab fragments are Fab fragments wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Bispecific antibody formats comprising crossover Fab fragments have been described, for example, in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010/136172, WO2010/145792 and WO2013/026831. The native Trastuzumab sequences has been optimized in their CDRs to improve the stability of the antibody CDRs against spontaneous chemical modification, the resulting sequences framework-grafted to avoid mispairing, and the bispecific antibody glycoengineered, resulting in highly potent bispecific antibodies that specifically bind to HER2 with enhanced FcgRIII binding resulting in enhanced recruitment of immune effector cells such as NK cells or monocytes/macrophages; finally they can be produced with high yield and only low percentage of side products comparable to the conventional parental Her2 antibodies. In the case of the HER2 bispecific CrossMAb antibody chain misparing of light chains resulting from the fact that both pertuzumab and trastuzumab are based on a comparable framework region has been overcome by grafting the CDRs on a completely novel antibody framework.
In another aspect of the invention monovalent bispecific antibodies specifically binding to the extracellular domains IV and II of HER2 are provided wherein the two binding moieties comprise identical light chains based on a consensus of the parental trastuzumab and pertuzumab light chains and the corresponding pertuzumab heavy chain has been remodeled. The use of this so-called ‘common light chain’ principle, i.e. combining two binders that share one light chain but still have separate specificities, prevents light chain mispairing and in this particular case retains the epitope specificity of the parental antibodies. As a consequence, there are less side products during production, facilitating the homogenous preparation of HER2 bispecific antigen binding molecules at high yields. Surprisingly the inventors of the present invention found that the bispecific HER2 antibodies in the monovalent common light chain format have an increased affinity to the pertuzumab epitope, and show superior inhibitory effects on cell proliferation and induction of cell dependent cytotoxicity (CDC) as compared to the combination of the parental antibodies. Complement dependent cytotoxicity (CDC) is very important for the optimal therapeutic monoclonal antibodies (mAb) function and is totally conserved even after a chemotherapy treatment. However, this activity is generated by some antibodies but not all of them.